Increasing attention has been focused on the delivery of genes as therapeutic agents (i.e., gene therapy) for the treatment of gene-associated diseases. In particular, researchers have been studying the use of non-viral methods of delivery, due to safety concerns with using potentially infectious viruses. One of the most promising non-viral methods in gene therapy is the use of cationic lipids as a delivery vehicle (Felger, Proc. Natl. Acad. Sci., 84:7413-7417, 1987). The cationic lipids bind with negatively charged DNA and facilitate entry of the DNA into target cells. Successful delivery of genes via lipids into airway epithelia of rodents (Hyde, Nature, 362:250-255, 1993) have been reported. The gene of interest is generally incorporated into plasmids which can be produced and isolated from bacterial cells. Cationic lipids are generally synthesized chemically or isolated from natural sources using various methods known in the art. If lipid delivery of genes proves to be effective, huge quantities of plasmids will be required for future gene therapy. However, current methods for producing genes may impede progress in this field.
Currently there are very few reports on plasmid yield enhancement. One method involves the addition of a protein inhibiting drug, chloramphenicol, into a host culture (Maniatis et al. Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press, 1989). This approach does not increase plasmid yield significantly and, in addition, chloramphenicol is toxic to humans (AHFS Drug Information, American Society of Hospital Pharmacists, 1994). In another method, the plasmid producing cells are grown in a fed batch mode (Hofman et al. , J. Basic Microbiol., 30(1):37-41, 1990). While this approach can produce large quantities of plasmids, production yield is only proportional to cell mass.
A need exists,to develop economically viable methods for producing plasmids on a large scale to meet the increasing demand.